West Nile virus (WNV) is a mosquito borne infectious agent that causes febrile illness and occasionally encephalitis. Outbreaks had been reported in Africa, Asia and Europe since 1937. In 1999, the first case of WNV was detected in New York City. Since establishing in North America, the virus has spread rapidly westward and southward resulting in the largest epidemics of neuorinvasive WNV disease ever reported. Thus, there is a need for vaccines and antivirals agents. Currently, there is no vaccine for human use or antivirals that are available to treat WNV infections, and the care is mainly supportive. WNV belongs to the family a flavivirus. Members of this family include dengue virus, yellow fever, Japanese encephalitis virus, St. Louis encephalitis virus, and tick borne encephalitis virus. These infections have a similar clinical presentation, which makes diagnosis of the causative agent only possible with sophisticated laboratory analysis which is time consuming and costly. Therefore, an antiviral would ideally demonstrate broad antiviral activity against flaviruses. Antivirals with activity against WNV are in various states of clinical development. The identified chemical compounds are believed to target the virus polymerase, NTPase/helicase and protease. Interestingly, these lead compounds also have activity against other members of the family flaviridae. Our hypothesis is the high degree of sequence similarity amongst the flaviviruses results in a similar viral replication in the host cell. A corollary to this hypothesis is that compounds that have activity against one flavivirus, WNV, will have potential as a broad spectrum anti-viral against the flaviviruses. If the hypothesis is correct, then this would mean that the probes generated would be useful to dissect similar pathways and assist in additional target development. If the hypothesis is incorrect and some compounds are broadly active and others are not, then the probes can be used to discriminate difference between the flaviviruses and learn more about WNV. We have developed a primary HTS assay that has validated against a 12,000 compounds in duplicate for anti-viral activity against WNV. In this proposal, we propose to utilize this assay to screen compounds for activity against WNV. Specifically we will: (Aim 1) Perform a primary screen using the MLPCN library to identify lead compounds with antiviral activity against WNV and (Aim 2) Perform secondary screens to determine the extent to which the active leads demonstrate specificity and selectivity. We will (Subaim 2A) perform dose response studies to determine the extent of a compounds efficacy and toxicity; (Subaim 2B) confirm lead compounds specific antiviral activity in a plaque assay; (Subaim 2C) confirm lead compounds specificity and breadth by determining the extent of the antiviral activity against a flaviviruses yellow fever. (Subaim 2D) perform confirmatory chemistry. PUBLIC HEALTH RELEVANCE: The goal of this project is to identify probes with anti-viral activity against West Nile virus (WNV) some of which will be broadly reactive against the flavivirdae viruses. The probes generated would be useful to dissect similar pathways and assist in additional target development. West Nile virus (WNV) is a mosquito borne infectious agent that causes febrile illness and occasionally encephalitis. Outbreaks had been reported in Africa, Asia and Europe since 1937. In 1999, the first case of WNV was detected in New York City. Currently, there is no vaccine for human use or antivirals that are available to treat WNV infections, and the care is mainly supportive. WNV belongs to the family a flavivirus. Members of this family include dengue virus, yellow fever, Japanese encephalitis virus, St. Louis encephalitis virus, and tick borne encephalitis virus. These infections have a similar clinical presentation, which makes diagnosis of the causative agent only possible with sophisticated laboratory analysis which is time consuming and costly. Therefore, an antiviral would ideally demonstrate broad antiviral activity against flaviruses. [unreadable] [unreadable] [unreadable]